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Biomedical subjects

G Ravagnan

Publications and source records attributed to G Ravagnan.

At least 19 recordsLinked to original sources
PubMed1992

Membrane aging during cell growth ascertained by Laurdan generalized polarization.

The sensitivity of the fluorescent probe Laurdan to the phase state of lipids has been utilized to detect modifications in the composition and physical state of cell membranes during cell growth. In phospholipid vesicles, the Laurdan emission spectrum shows a 50-nm red shift by passing from the gel to the liquid-crystalline phase. The Generalized Polarization (GP) value has been used for the data treatment instead of the ratiometric method common in investigations utilizing other fluorescent probes that display spectral sensitivity to medium properties. The GP value can be measured easily and quickly and possesses all the properties of "classical" polarization, including the additivity rule. Once Laurdan limiting GP values have been established for the gel and the liquid-crystalline phase of lipids, the quantitative determination of coexisting phases in natural samples is possible. In the present work the observation of a relevant decrease in the fractional intensity of the liquid-crystalline phase in K562 cell membranes during 5 days of asynchronous growth is reported. A decrease in the "fluidity" of cell membranes in K562 cells kept in culture for several months is also reported. The procedure developed for labeling cell membranes with Laurdan is reported and the influence of cell metabolism on fluorescence parameters is discussed. Also discussed is the influence of cholesterol on Laurdan GP.

2-Naphthylamine↗
PubMed1992

Membrane oxidative damage induced by ionizing radiation detected by diphenylhexatriene fluorescence lifetime distributions.

The sensitivity of the fluorescence lifetime of 1,6-diphenyl- 1,3,5-hexatriene (DPH) to the dielectric constant of its environment has been used to detect oxidative damage to phospholipid membranes induced by ionizing radiation. The DPH fluorescence decay in phospholipid vesicles is described well by a continuous distribution of lifetime values, reflecting the various DPH depths in the bilayer and related to the gradient of the dielectric constant. Ionizing radiation oxidizes unsaturated acyl residues of phospholipids, altering the dielectric constant across the bilayer, sharpening the distribution of DPH lifetimes and increasing the centre of the distribution. Ionizing radiation doses between 22 and 110 Gy were used, and were effective only in the presence of oxygen. A model based on the formation of packing defects in the bilayer describes the phenomenon.

Diphenylhexatriene↗
PubMed1992

Charge and pH effect on the early events of Epstein-Barr virus fusion with lymphoblastoid cells (Raji).

Fusion of Epstein-Barr virus (EBV) with Raji cells was measured after exposure of the virus to neutral or low pH, enzymatic modification of the viral spike glycoproteins, or chemical modification of the target membrane. The relief of octadecylrhodamine (R18) fluorescence self-quenching was used to monitor fusion. Fusion of EBV with Raji cells at pH 5.9 was significantly enhanced compared to that at neutral pH. Treatment of Raji cells with agents known to modify the surface net charge (trinitrobenzene sulfonic acid) totally prevented fusion at a neutral pH. Desialylation of EBV significantly reduced the extent of fusion with Raji cells. Our results demonstrate that EBV is rapidly internalized and then fuses with lymphoblastoid cells in the endocytic vesicles.

Cells, Cultured↗
PubMed1991

Size and stability of dipalmitoylphosphatidylcholine/cholesterol unilamellar vesicles are affected by interaction with proteins.

The effect of entrapping the enzyme ascorbate oxidase into dipalmitoylphosphatidylcholine/cholesterol vesicles, was studied by conventional transmission electron microscopy and freeze-fracture. The freeze-fracture technique has definitely demonstrated the unilamellar nature of empty and enzyme-loaded vesicles. Images of freeze-fractured and label-fractured liposomes also indicate that the observed reduction of vesicles volume could be related to the localization of ascorbate oxidase across the membrane. The membrane localization of ascorbate oxidase may explain the oxidation of externally added ascorbate by intact enzyme-loaded liposomes. Finally, the ageing of liposomes appears to be accelerated in the presence of proteins.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1991

Quantitation of lipid phases in phospholipid vesicles by the generalized polarization of Laurdan fluorescence.

The sensitivity of Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) excitation and emission spectra to the physical state of the membrane arises from dipolar relaxation processes in the membrane region surrounding the Laurdan molecule. Experiments performed using phospholipid vesicles composed of phospholipids with different polar head groups show that this part of the molecule is not responsible for the observed effects. Also, pH titration in the range from pH 4 to 10 shows that the spectral variations are independent of the charge of the polar head. A two-state model of dipolar relaxation is used to qualitatively explain the behavior of Laurdan. It is concluded that the presence of water molecules in the phospholipid matrix are responsible for the spectral properties of Laurdan in the gel phase. In the liquid crystalline phase there is a relaxation process that we attribute to water molecules that can reorientate during the few nanoseconds of the excited state lifetime. The quantitation of lipid phases is obtained using generalized polarization which, after proper choice of excitation and emission wavelengths, satisfies a simple addition rule.

2-Naphthylamine↗
PubMed1991

Alterations in erythrocyte membrane lipids induced by low doses of ionizing radiation as revealed by 1,6-diphenyl-1,3,5-hexatriene fluorescence lifetime.

Damage in membrane lipids induced by low doses of ionizing radiation in the presence of oxygen has been detected in rabbit erythrocyte ghosts labelled with 1,6-diphenyl-1,3,5-hexatriene (DPH). Multifrequency phase and modulation fluorometry was used to measure DPH fluorescence lifetime. This technique is particularly suited for the observation of heterogeneous fluorescence decays. DPH decay in erythrocyte membranes is described by a two-component continuous distribution of lifetimes. The value of the distribution width of the long-lived component is found to be affected by radiation-induced membrane lipid damage at doses as low as 0.5 Gy, well within the dose range used to measure cell survival. The width of the DPH lifetime distribution decreases when the ghosts are irradiated in the presence of oxygen. Such a decrease is a linear function of the logarithm of the dose. After a dose of 110 Gy and above, the fractional intensity of the short-lived component of the DPH decay increases linearly, indicating severe membrane damage. Experiments performed in the absence of oxygen do not show any change in the fluorescence parameters up to a dose of 550 Gy. The molecular identification of the produced damage has not been accomplished, but the necessity of oxygen to observe the damage suggests that hydroperoxides and lipids crosslinks are produced.

Animals↗
PubMed1991

Enterosistem 18-R: description and comparative evaluation with conventional methods for identification of members of the family Enterobacteriaceae.

The efficiency and accuracy of Enterosistem 18-R (Liofilchem s.r.l., Roseto degli Abruzzi, Teramo, Italy) were compared with those of conventional biochemical methods to identify 360 members (38 species) of the family Enterobacteriaceae. Overall, 329 strains (91.3%) were correctly identified (percentage of identification, greater than or equal to 90.0), with 37 (11.2%) requiring additional tests for complete identification. For 11 isolates (3.1%), Enterosistem 18-R gave only genus identifications, and for 14 (3.9%), the strains did not correspond to any key in the codebook and could not be identified by the manufacturer's computer service. Only six isolates (1.7%) were misidentified. The new system accurately identified common and several newly described isolates of the family Enterobacteriaceae, such as Enterobacter gergoviae, Providencia rustigianii, Serratia odorifera, and Serratia rubidaea. The system is highly reproducible, simple to perform, easy to handle, and inexpensive. With adjustments in supplementary code numbers for some strains, Enterosistem 18-R is a suitable alternative for identification of members of the Enterobacteriaceae in clinical laboratories.

Bacteriological Techniques↗
PubMed1990

In vivo and in vitro inhibition of platelet aggregation by SV-IV, a major protein secreted from the rat seminal vesicle epithelium.

In summary, the present study documents that platelet aggregation triggered by thrombin, ADP, collagen and PAF both in vivo and in vitro, was prevented by SV-IV in a dose-dependent manner. Only platelet aggregation by AA was not affected by the protein, thus suggesting a possible involvement of PLA2 inhibition in the molecular mechanism at the basis of SV-IV anti-thrombotic effect.

Animals↗
PubMed1989

Immunosuppressive and anti-inflammatory properties of a major protein secreted from the epithelium of the rat seminal vesicles.

The nonspecies specific immunosuppressive and anti-inflammatory properties of a major protein (SV-IV) secreted from the epithelium of rat seminal vesicles (SV) are described. To detect the immunosuppressive effect, peripheral blood lymphocytes (PBL) were pretreated for 2 hr at 37 degrees with SV-IV, and the protein was maintained in the incubation medium during the whole culture time. We obtained evidence that, during preincubation of PBL and SV-IV the protein was transformed by a transglutaminase (TGase) released from PBL into modified low and high molecular weight forms able to bind to PBL surfaces. It is suggested that T lymphocytes are the possible targets of the immunosuppressive effect. SV-IV seems to inhibit only the early phase of the proliferative response of T lymphocytes to mitogens without having any direct effect on the enzymatic system involved in DNA synthesis. Moreover, the protein SV-IV was also shown to possess an anti-inflammatory property due to a block of the arachidonic acid cascade at the level of the enzyme phospholipase A2 (PLA2). The physiological significance of the immunosuppressive and anti-inflammatory properties of SV-IV are discussed in relation to different aspects of the mammalian reproduction.

Animals↗
PubMed1989

Inhibitory effect of SV-IV, a major protein secreted from the rat seminal vesicle epithelium, on phagocytosis and chemotaxis of human polymorphonuclear leukocytes.

The effect of SV-IV, one of the major proteins secreted from the rat seminal vesicle epithelium, on phagocytosis and chemotaxis of human polymorphonuclear leukocytes (PMNs) has been studied. Various cytological, biochemical, metabolic, and physical correlates of both biological activities have been found to be markedly reduced by the presence in the medium of micromolar concentrations of protein SV-IV. Moreover, the Scatchard analysis of the labeled SV-IV binding to PMN cell surface has demonstrated that such binding is specific. The binding sites contain only saturable components, completely displaceable by unlabeled SV-IV. The number of the specific sites has been calculated to be 87,000/cell, with a Kd of 1.72 X 10(-7) M. The molecular mechanism of the inhibitory effect is discussed along with the possible biological and clinical implications of the experimental findings.

Animals↗
PubMed1989

Inhibition of macrophage phagocytic activity by SV-IV, a major protein secreted from the rat seminal vesicle epithelium.

The protein SV-IV, one of the major secretory proteins produced by the rat seminal vesicle epithelium, has been found to possess a marked ability to inhibit in vitro the phagocytic properties of activated peritoneal rat macrophages, by a mechanism that apparently involves phagocytes and target cells. Although SV-IV is a substrate for transglutaminase (TGase), an enzyme secreted by activated macrophages, TGase does not seem to play any significant role either in the binding of the protein to the cells participating in the phagocytic process or in the inhibition of macrophage phagocytosis by SV-IV. The significance of the findings in relation to the reproductive process and their possible clinical implications are discussed.

Animals↗
PubMed1989

Glutathione transferase in bacteria: subunit composition and antigenic characterization.

The presence of glutathione transferase (GST; EC 2.5.1.18) in Escherichia coli ATCC 25922, E. coli ATCC 25422, Proteus vulgaris ATCC 8427, Pseudomonas aeruginosa ATCC 27853, Klebsiella oxytoca CIP 666, K. oxytoca AF 101, Enterobacter cloacae CIP 6085, Serratia marcescens CIP 6755, and Proteus mirabilis AF 2924 was investigated. Using 1-chloro-2,4-dinitrobenzene as substrate, GST activity was found in the glutathione-(GSH-)affinity-purified fraction of all strains tested. SDS-PAGE analysis of GSH-affinity-purified enzyme indicated that the GSTs of all these bacteria are dimers of two identical subunits of Mr about 22,500. Rabbit antiserum directed against the major isoenzyme present in Proteus mirabilis AF 2924, Pm-GST-6.0, was used to investigate the antigenic properties of bacterial GSTs. Western blot analysis indicated that a GST antigenically identical to Pm-GST-6.0 is present in Enterobacter cloacae CIP 6085, Escherichia coli ATCC 25422 and Proteus vulgaris ATCC 8427, but absent in Escherichia coli ATCC 25922, Klebsiella oxytoca CIP 666, K. oxytoca AF 101 and Serratia marcescens CIP 6755. The presence of Pm-GST-6.0, but not mammalian GST, increased the MIC values of amikacin, ampicillin, cefotaxime, cephalothin and nalidixic acid for E. coli ATCC 25922. It is suggested that bacterial GST may represent a defense against the effects of antibiotics.

Anti-Bacterial Agents↗
PubMed1989

In vitro activity of netilmicin alone and in combination with azlocillin, mezlocillin and imipenem against 149 coagulase-negative staphylococci.

Coagulase-negative staphylococci (CNS) have long been regarded as innocuous skin commensals with little pathogenic potential but they have recently become, under appropriate conditions, an important cause of infections. In fact, infections caused by CNS are an increasing problem especially, but not exclusively, in immuno-compromised patients. A total of 149 strains of CNS were identified from 47 patients admitted to the Haematology Department of Pescara Hospital from October 1986 to November 1987. The strains, isolated from different parts of the body and characterized by their methicillin susceptibility, were classified by API-Staph in 11 different groups. MICs and MBCs of netilmicin alone and combined with azlocillin, mezlocillin and imipenem were studied. For all combinations FIC and FBC indices were determined. The killing kinetics of the drugs mentioned above were also determined. Except for a few microorganisms (less than 5%), the associations showed a synergic or additive effect.

Azlocillin↗
PubMed1988

Purification and characterization of three forms of glutathione transferase from Proteus mirabilis.

Three forms of glutathione transferase (GST) with pI values of 6.0, 6.4 and 7.3 were isolated from Proteus mirabilis AF 2924 by glutathione-affinity chromatography followed by isoelectric focusing, and their structural, kinetic and immunological properties were investigated. Upon SDS/polyacrylamide-slab-gel electrophoresis, all forms proved to be composed of two subunits of identical (22,500) Mr. GST-6.0 and GST-6.4 together account for about 95% of the total activity, whereas GST-7.3 is present only in trace amounts. Extensive similarities have been found between GST-6.0 and GST-6.4. These include subunit molecular mass, amino acid composition, substrate specificities and immunological characteristics. GST-7.3 also cross-reacted (non-identity) with antisera raised against bacterial GST-6.0. None of the antisera raised against a number of human, rat and mouse GSTs cross-reacted with the bacterial enzymes, indicating major structural differences between them and the mammalian GSTs. This conclusion is further supported by c.d. spectra.

Amino Acids↗